The objectives of the present project were to set up, optimise and characterise a digitalholographic microscopy (DHM) laboratory set-up designed for the study of eyetissue and to implement and optimise digital data processing and noise reductionroutines. This work is part of a collaborative project aiming to provide quantitativemethods for the in vitro and in vivo characterisation of human corneal transparency.The laboratory set-up is based on a commercial laboratory microscope with zoomfunction (a “macroscope”). In continuation of previous work, we completed and optimised,and extended a software for holographic signal processing and numericalpropagation of the wavefront.To characterise the set-up and quantify its performances for standard operationand in its DHM configuration, we compare the magnification and resolution to theoreticalvalues for a given set of parameters. We determined the magnification factorand the rotation angle between the object and camera planes. With a laser wavelengthof 532 nm, a x1 objective and a zoom setting of x2.9 (which corresponds to aplane sample wavefront), we measured a magnification of 1.68. With the same parameters,we measure a holographic resolution of about 11 m. The wavefront phasecould be determined with a precision of a fraction of the wavelength.We subsequently performed analysis of the relative contribution of coherent noiseand implemented and evaluated several noise reduction routines. While the impactof coherent noise remained visible in the amplitude image, interferometric precisionwas obtained for the phase of the wavefront and the set-up was considered qualifiedfor its intended use for corneal characterisation.A first test measurement was performed on primate cornea.Subsequent work will address the further quantitative characterisation of the setupfor the full set of parameters (objectives, zoom positions, wavelengths), test measurementson samples with known transmission and light scattering properties (e.g.solutions of PMMA beads) and the comparison of the results with the predictions ofa theoretical model, and measurements on animal and human tissue.

Moving towards label-free technologies is essential for many clinical and research applications. Raman spectroscopy is a powerful tool in the field of biomedicine for label-free cell characterisation and disease diagnosis, owing to its high chemical specificity. However, Raman scattering is a relatively weak process and can require long acquisition times, thus hampering its integration to clinical technologies. Multimodal analysis is currently pushing the boundaries in biomedicine, obtaining more information than would be possible using a single mode and overcoming any limitations specific to a single technique. Digital holographic microscopy (DHM) is a rapid and label-free quantitative phase imaging modality, providing complementary information to Raman spectroscopy, and is thus an ideal candidate for combination in a multimodal system. Firstly, this thesis explores the use of wavelength modulated Raman spectroscopy (WMRS), for the classification of immune cell subsets. Following this a multimodal approach, combining Raman spectroscopy and DHM, is demonstrated, where each technique is considered individually and in combination. The complementary modalities provide a wealth of information (both chemical and morphological) for cell characterisation, which is a step towards achieving a label-free technology for the identification of human immune cells. The suitability of WMRS to discriminate between closely related neuronal cell types is also explored. Furthermore optical spectroscopic techniques are useful for the analysis of food and beverages. The use of Raman and fluorescence spectroscopy to successfully discriminate between various whisky and extra-virgin olive oil brands is demonstrated, which may aid the detection of counterfeit or adulterated samples. The use of a compact Raman device is utilised, demonstrating the potential for in-field analysis. Finally, monodisperse and highly spherical nanoparticles are synthesised. A short study demonstrates the potential for these nanoparticles to benefit the techniques of surface enhanced Raman spectroscopy and optical trapping, by way of minimising variability.

The main objective of this thesis is the development of new processing techniques for digital holograms. The present work is part of the HoloFlow project that intends to integrate the DHM technology for the monitoring of water quality. Different tools for an automated analysis of digital holograms have been developed to detect, refocus and classify particles in continuous fluid flows. A detailed study of the refocusing criterion permits to determine its dependencies and to quantify its robustness. An automated detection procedure has been developed to determine automatically the 3D positions of organisms flowing in the experiment volume. Two detection techniques are proposed: a usual method based on a global threshold and a new robust and generic method based on propagation matrices, allowing to considerably increase the amount of detected organisms (up to 95 %) and the reliability of the detection. To handle the case of aggregates of particles commonly encountered when working with large concentrations, a new separation procedure, based on a complete analysis of the evolution of the focus planes, has been proposed. This method allows the separation aggregates up to an overlapping area of around 80 %. These processing tools have been used to classify organisms where the use of the full interferometric information of species enables high classifier performances to be reached (higher than 93 %). / Doctorat en Sciences de l'ingénieur / info:eu-repo/semantics/nonPublished

Práce se zabývá interpretací kvantitativního fázového zobrazení pomocí techniky koherencí řízené holografické mikroskopie. Vzhledem k tomu, že tato technika generuje velké množství kvantitativních fázových obrazů o nezanedbatelné velikosti, manuální analýza by byla časově náročná a neefektivní Za účelem urychlení analýzy obrazů získaných pomocí koherencí řízené holografické mikroskopie je v této práci navržena metodika automatizované interpretace kvantitativních fázových obrazů pomocí strojového učení s učitelem. Kvantitativní fázové obrazy umožňují extrakci parametrů charakterizujících distribuci suché hmoty v buňce a poskytují tak cennou informaci o buněčném chování. Cílem této práce je navrhnout metodologii pro automatizovanou klasifikaci buněk při využití této kvantitativní informace jak ze statických, tak z časosběrných kvantitativních fázových obrazů. Navržená metodika byla testována v experimentech s živými buňkami, jimiž byla vyhodnocena výkonnost klasifikace a významnost parametrů získaných z kvantitativních fázových obrazů.

The goal of this diploma thesis was using of a coherence-controlled holographic microscope in cell’s life research. A brief history of interference microscopy and it’s applications in biology is described. Also other microscopy techniques routinely used for transparent objects imaging are mentioned and the biology of cell’s life cycle briefly explained. Characteristics describing the shape of a cell were proposed and tested with respect to identification of particular phases of its life cycle. The method of dynamic phase differences was modified in order to distinguish the internal motion of cell’s mass from the movement of the whole cell. Selected characteristics were used to evaluate observations carried out with the holographic microscope and the possibilities of their further applications were depicted. In conclusion, obtained findings were summarized and modifications of microscope construction as well as data-processing software were suggested.

No description available.

The diploma thesis deals with the registration of images taken with the multimodal holographic microscope (MHM). The summary covers the fluorescent and holographic microscopy, and the multimodal holographic microscope combining both these microscopy types. Every pair of the images needs to be aligned in order to gain new information by combining both image types. The thesis contains an algorithm that registers images by phase correlation as well as a process created in MATLAB in accordance with the algorithm. The most important procedure parameters’ influence on the registration success is described and the results are annotated.

The subject of the PhD thesis is the application of a transmission digital holographic microscope (DHM) which was designed and constructed in the Laboratory of optical microscopy at the IPE BUT for the research of live cells dynamics. First part of the work is concerned with theoretical description of the microscope imaging properties dependent on the coherence of illumination. It is supplemented with experiments of imaging of a model and a real biological specimen. The following part describes construction modifications and innovations of the microscope and its equipment that enabled the utilization of the microscope for live cells observations. In the experimental part the methodology of live cells preparation and DHM imaging was worked out. The methodology was verified by the observation of cell dynamics during an apoptosis induced by the cytostaticum cis-platinum. Further experiments examined the dynamics of live cells in standard conditions and during a deprivation stimulus. A novel method of holographically reconstructed phase, named \uva{dynamic phase differences}, was set up to evaluate quantitative changes of cell mass distribution during the experiments. Depending on the degree of malignancy and density of cell outgrowth, various schemes of cancer cells behaviour during a specific reaction were revealed using this method. For the quantitative analysis of the DHM phase imaging, a suitable statistical characteristic and an interpretation of the measured data were proposed. Both of them were successfully applied for the comparison of cell motility of two cell types: parental and progeny cell lines. On the basis of the proposed processing, hypotheses describing the reaction mechanism of tumour cells to stress life conditions were established. In the conclusions we summarize our findings and suggestions for the construction and the applications of a new generation of the transmission DHM.

This dissertation thesis deals with the methods for processing image data in X-ray microtomography and digital holographic microscopy. The work aims to achieve significant acceleration of algorithms for tomographic reconstruction and image reconstruction in holographic microscopy by means of optimization and the use of massively parallel GPU. In the field of microtomography, the new GPU (graphic processing unit) accelerated implementations of filtered back projection and back projection filtration of derived data are presented. Another presented algorithm is the orientation normalization technique and evaluation of 3D tomographic data. In the part related to holographic microscopy, the individual steps of the complete image processing procedure are described. This part introduces the new orignal technique of phase unwrapping and correction of image phase damaged by the occurrence of optical vortices in the wrapped image phase. The implementation of the methods for the compensation of the phase deformation and for tracking of cells is then described. In conclusion, there is briefly introduced the Q-PHASE software, which is the complete bundle of all the algorithms necessary for the holographic microscope control, and holographic image processing.

The development of techniques targeting the manipulation of particles of different<p>sizes - mostly in the nano to millimeter scale - when dispersed in a carrier medium, is an increasingly important topic in many fields such as biotechnology,nanotechnology, medicine, biophysics and environmental monitoring and remediation. The underlying rationale for using such techniques stands in the sometimes compelling requirements of avoiding clogging as in micro/nano channel flows, of limiting sedimentation and wall interactions in particle/cell counting, of enhancing particle-surface interaction as in bio-sensing or of facilitating characterization and sorting as in bio-physical applications. Being developed in the frame of a Belgian national project devoted to the characterization and counting of pollutant in water media by digital holographic microscopy, this thesis tackles a peculiar class of particle manipulation techniques, commonly known as Focusing. The main goal of focusing is to avoid at best wall particle interactions and sedimentation, prevalent issues for dispersions owing in micro/mini-channels especially for applications such as optical characterization and counting.<p><p>The main attention was given to two flow focusing techniques - Hydrodynamic and Acoustic Focusing - for their wide range applicability and cost effectiveness. Hydrodynamic Focusing consists in controlling the position and spreading of the sample under investigation by means of a so-called sheath flow. A low-cost, nevertheless effective, prototype has been conceived, designed, manufactured and tested. It allowed for controlling the spreading of the sample stream and achieving a focusing ratio accounting for only 4% of the original stream width.<p><p>Acoustic Focusing takes advantage of the time-averaged pressure fields induced by the creation of standing waves in channels to manipulate and focus the dispersed particles. In the frame of this thesis, several devices have been developed using square cross section glass mini-channels. Aside from the cost-effectiveness, particles where focused in a somehow unexpected but high reproducible 3D matrix-like structure. A novel numerical model has also been implemented in order to study the conditions leading to the 3D structure formation. A good agreement between experimental and numerical results was found./Ce projet de thèse portant sur la manipulation de micro-particules dans des minicanaux s'inscrit dans le développement de cellules de flux pour des applications biologiques, qui est l'une des problématiques du projet HOLOFLOW, soutenu par<p>la région de Bruxelles Capitale. Les cellules de flux doivent permettre l'observation et la reconnaissance des micro-organismes vivants dans une large gamme de dimensions (de quelques microns à 1mm) avec la microscopie holographie digitale.<p>La problématique d'observation et de manipulation des microorganismes en flux est liée au clogging (bouchage) et à la sédimentation qui limitent la durée de vie des cellules d'observation. Ce projet de thèse s'inscrit dans cette problématique et propose deux axes d'étude pour limiter l'interaction entre organismes et canaux, la focalisation hydrodynamique, basée sur le guidage de flux, et la focalisation acoustique, basée sur la manipulation des particules.<p><p>La focalisation hydrodynamique est une technique basée sur l'injection différentiée de l'échantillon à observer et d'un fluide support. La différence des vitesses d'injection des flux permet de contrôler la dispersion des particules afin d'optimiser leur observation. Dans le cadre de cette thèse, un prototype à bas-coût a été développé et construit, permettant de focaliser les particules dans un faisceau jusqu'à 4% de leur faisceau incident.<p><p>La focalisation acoustique utilise la création d'une onde acoustique stationnaire afin de regrouper les particules en suspension au centre du canal. Au cours de cette thèse, plusieurs prototypes ont été réalisés, mettant en évidence la formation de motifs tridimensionnaux. Un model numérique a été spécialement développé afin d'étudier les conditions de génération de ces motifs, et de nombreuses expériences ont été menées afin de s'assurer de leur reproductibilité. Une bonne adéquation entre la position des particules mesurée et calculée numériquement a été démontrée. / Doctorat en Sciences de l'ingénieur / info:eu-repo/semantics/nonPublished